This in vitro cell-based study identified the contributing role of pyocyanin in the resistance of Pseudomonas aeruginosa to ciprofloxacin. Problem statement: P. aeruginosa is the major pathogen in the Cystic Fibrosis (CF) lung with pyocyanin being a critical component of its virulence. Prevalence is high and, once acquired, chronic infection is difficult to eliminate. Ciprofloxacin remains a crucial oral agent effective against P. aeruginosa, but resistance is increasingly reported. Approach:Here we examined the extent to which exogenously added pyocyanin affected P. aeruginosa susceptibility to ciprofloxacin and the contribution of altered efflux activity and biofilm production with the aim of ultimately increasing sensitivity. Results: Metabolic conversion of resazurin to resorufin was used as an index of bacterial cell growth while fluorescent measurement of acriflavine efflux and crystal violet staining was used as markers of efflux activity and biofilm production, respectively. Pyocyanin (100 卩 added exogenously decreased susceptibility of two P. aeruginosa strains, PAO1 and ATCC 27853 to ciprofloxacin at 125 and 500 mg L-1, respectively. Exogenously added pyocyanin decreased efflux activity in both strains while biofilm production was significantly increased. Conclusion: We conclude that increased biofilm production may contribute to the observed decreased susceptibility of P. aeruginosa to ciprofloxacin. Ciprofloxacin is a crucial orally effectively agent against P. aeruginosa with resistance being increasingly reported. This initial study highlights a potential mechanism that may underlie this resistance which may be of clinical interest. Further studies using additional antibiotics and the pyocyanin precursor 1-hydroxyphenazine are required.